In general this invention relates to a coated substrate and methods of making the coated substrate. Specifically, the invention is a substrate having a charged film for use in the optical, electrical and biological fields, and a method of making the substrate having a charged film.
Analysis of the structure, organization and sequence of nucleic acid molecules is of profound importance in the prediction, diagnosis and treatment of human disease and in the study of gene discovery, expression, and development. A fundamental tool used in the analysis of nucleic acid molecules is the high density array (HDA). The HDA provides the framework for the immobilization of nucleic acid molecules for analysis on a rapid, large-scale basis. HDAs generally include a substrate having a large number of positionally distinct DNA probes attached to a surface of the substrate for subsequent hybridization to a DNA target. The key to efficiently immobilizing nucleic acid molecules is the surface chemistry and surface morphology of the HDA substrate.
The surfaces of both organic and inorganic substrates can be modified by the deposition of a polymer monolayer coating or film in order to construct biomolecular assemblies. In addition, surface modification can also be used to promote adhesion and lubrication, prevent corrosion, modify the electrical and optical properties of the substrate surface, and create electroactive films suitable for various optical and electronic sensors and devices.
However, the problem with monolayer films is that they were not capable of being modified to achieve a desired surface characteristic such as uniform electrical charge with a precisely controlled thickness and molecular organization. In order to obtain this desired surface characteristic, a multilayered film having a well-defined molecular organization in a smooth geometrical arrangement was produced on a substrate. Two methods that were developed to achieve this goal for the preparation of multilayered films were the Langmuir-Blodgett method and the Decher et al. method.
In the Langmuir-Blodgett (LB) technique, a monolayer is formed on the surface of water and then transferred onto solid substrates. Paras N. Prasad and W. M. K. P. Wijekoon, Langmuir-Blodgett Films (Organic Polymers for Photonics Applications) pp. 3552-3554 (Joseph C. Salamone, POLYMERIC MATERIALS ENCYCLOPEDIA (1996)). The following steps show how the complicated LB technique is performed. First, a molecular structure with amphiphilic properties is dissolved in an appropriate solvent and spread on the surface of water. Second, the molecules with amphiphilic properties on the surface of the water are compressed slowly to form a compact monolayer by monitoring the surface pressure-area isotherm. Third, the monolayer is transferred from the water surface onto the surface of a substrate while under the pressure-area isotherm by insertion into or withdrawal of the substrate from the floating monolayer.
Although this process is effective for depositing multilayered films, it is subject to limitations. For example, the substrates to be coated by the LB method are limited in size due to the required mechanical manipulation in the film building process. Further, the films produced by this method are subject to gaps or spaces due to the non-uniform close packing of the amphiphiles on the liquid surface before the transfer onto a the solid substrate.
Another disadvantage of films created by the LB technique is that they are inherently unstable. The instability is due to the transfer of the films to the substrate in liquid form, which allows for adhesion of the film to the substrate primarily through weak van der Waals forces; therefore, the layers of the LB film that constitute the surface of the substrate are mechanically unstable.
Decher et al., U.S. Pat. No. 5,208,111, incorporated for all purposes, herein by reference, has demonstrated that it is possible to build up multilayer thin films onto charged surfaces via alternating deposition of polycations and polyanions. (See also Decher, Science. 277, 1233 (1997). The Decher method is illustrated by FIGS. 1A-1F.
FIG. 1A shows a schematic of the film deposition process as one slide 10 is progressively dipped into a series of beakers. Beaker 1 contains a polyanion solution; beaker 2 contains a wash solution, beaker 3 contains a polycation solution, and beaker 4 also contains a wash solution. The slide 10 having a layer of cations attached to its surface is dipped sequentially into beakers 1-4. After being removed from beaker 1, the slide 10 contains a monolayer polyanionic coating electrostatically attached to the cation layer. It retains this coating after washing in beaker 2. The slide 10 next receives a polycationic monolayer coating electrostatically attached to the polyanion monolayer by dipping in beaker 3. After washing in beaker 4, a single polyanion/polycation bilayer is attached to the prior oppositely charged layer of the slide. The process may be repeated for any number of polyanion/polycation bilayers. The four steps are the basic buildup sequence for the simplest bilayer film, (A/B)n formed by the attachment of a polyanion to a polycation.
The construction of more complex films may be accomplished by using different polycation/polyanion solutions.
FIGS. 1B to 1D are a simplified molecular picture of the first two adsorption steps as illustrated in FIG. 1A, depicting film deposition starting with a slide 10. Counterions were omitted for clarity. FIG. 1B depicts the slide 10 prior to immersion into the polyanion solution 2. The slide 10 has a cation layer 12 on the surface. The polyion conformation and layer interpenetration represent an idealization of the surface charge reversal with each adsorption step. FIG. 1C depicts the slide 10 after removal from beaker 2. The cation layer 12 has a polyanion layer 14 attached to it. FIG. 1D depicts the substrate 10 after removal from beaker 4. Finally, the polyanion layer 14 is attached to a polycation layer 16.
FIG. 1E represents the chemical structure of a typical polyanion, the sodium salt of poly(styrene sulfonate) (PSS) 18, used in the Decher method. FIG. 1F represents the chemical structure of a typical polycation, poly(allylamine hydrochloride) (PAH) 20.
Using the Decher method, in the post-preparation treatment of the films, the surface is smoothed by consecutive immersions of the films in solutions of salt. Presumably, the salt breaks some of the anion-cation bonds, and their removal by washing in pure water leads to the reformation of the polymer chains in a more equilibrated conformation. The resulting smooth film creates a problem for use as a substrate for performing DNA hybridization experiments, viz. that a single stranded DNA molecule is more likely to become physically pinned down due to electrostatic interactions along the entire length of the smooth charged substrate surface. This xe2x80x9cpinningxe2x80x9d effect prevents free access by a complimentary strand, and thereby inhibits hybridization.
The present invention is a multilayered charged thin film that has an inherent and controlled surface roughness that has been demonstrated to facilitate and enhance the immobilization and hybridization of nucleic acids. The present invention is a reproducible, simple and effective procedure for making multilayered charged thin films. Once coated by the disclosed method, a substrate will facilitate the improved retention of nucleic acids to its surface by ionic interactions. This multilayered charged thin film on a substrate forms a durable surface that will endure the effect of time and adverse environmental conditions. This invention provides a way to obtain homogeneous, less labile and more resistant thin films compared with the xcex3APS aminated and other positively charged surfaces.
The present invention is a multilayered charged thin film applied to a substrate with a surface charge. A first layer of polyelectrolyte having an opposite charge to the substrate surface charge adheres to the substrate electrostatically. Additional polyelectrolyte layers can be placed on top of the first polyelectrolyte layer as long as additional layers have an opposite charge from the charge of the prior layer. In order to achieve a desired roughness, each successive layer is deposited in different solutions of an alternatively charged polyelectrolyte mixed with salt. The polyelectrolyte layers are composed to achieve a precise surface roughness that optimizes the adhesion of a binding entity and facilitates the hybridization of DNA in performing DNA hybridization assays. The final polyelectrolyte layer is aminated or activated for noncovalent bonding to a specific binding entity.